Super‐Resolution Mapping of Single Nanoparticles inside Tumor Spheroids

Cancer spheroids have structural, functional, and physiological similarities to the tumor, and have become a low-cost in vitro model to study the physiological responses of single cells and therapeutic efficacy of drugs. However, the tiny spheroid, made of a cluster of high-density cells, is highly scattering and absorptive, which prevents light microscopy techniques to reach the depth inside spheroids with high resolution. Here, a method is reported for super-resolution mapping of single nanoparticles inside a spheroid. It first takes advantage of the self-healing property of a "nondiffractive" doughnut-shaped Bessel beam from a 980 nm diode laser as the excitation, and further employs the nonlinear response of the 800 nm emission from upconversion nanoparticles, so that both excitation and emission at the near-infrared can experience minimal loss through the spheroid. These strategies lead to the development of a new nanoscopy modality with a resolution of 37 nm, 1/26th of the excitation wavelength. This method enables mapping of single nanoparticles located 55 microm inside a spheroid, with a resolution of 98 nm. It suggests a solution to track single nanoparticles and monitor their release of drugs in 3D multicellar environments.